Specific pathogen-free macaques: definition, history, and current production

Specific pathogen-free (SPF) macaque colonies are now requested frequently as a resource for research. Such colonies were originally conceived as a means to cull diseased animals from research-dedicated colonies, with the goal of eliminating debilitating or fatal infectious agents from the colony to improve the reproductive capacity of captive research animals. The initial pathogen of concern was Mycobacterium tuberculosis (M.tb.), recognized for many years as a pathogen of nonhuman primates as well as a human health target. More recently attention has focused on four viral pathogens as the basis for an SPF colony: simian type D retrovirus (SRV), simian immunodeficiency virus (SIV), simian T cell lymphotropic/leukemia virus (STLV), and Cercopithecine herpesvirus 1 (CHV-1). New technologies, breeding, and maintenance schemes have emerged to develop and provide SPF primates for research. In this review we focus on the nonhuman primates (NHPs) most common to North American NHP research facilities, Asian macaques, and the most common current research application of these animals, modeling of human AIDS.     

B-virus specific-pathogen-free breeding colonies of macaques (Macaca mulatta): retrospective study of seven years of testing.

BACKGROUND AND PURPOSE: National Institutes of Health's Division of Comparative Medicine has sponsored a multi-institutional program for the establishment of specific-pathogen-free (SPF) macaque colonies. B virus (Herpesvirus simiae, Cercopithecine herpesvirus type 1) has been targeted in this surveillance. Participating institutions have established individual timetables for frequency of testing and types of monitoring and husbandry techniques, all with the common goal of producing pathogen-free monkeys for research. The greatest biosecurity threat to the program comes from failure to detect seronegative latent infections, either in first-year macaques or macaques introduced in subsequent years, although these are supposed to operate as closed colonies. METHODS: From January 1990 through December 1996, we screened macaques for B virus, using enzyme-linked immunoabsorbant assay (ELISA) and Western blot analysis. RESULTS: During the first year, 1,097 macaques from six colonies were tested, and 88.4% tested negative for B virus. During the seventh year, 1,843 were tested, of which 99.7% tested negative. Seropositive macaques were detected as late as the seventh year. CONCLUSIONS: An aggressive program to establish an SPF colony of captive breeding macaques can be effective in reducing the risk of B-virus exposure.     

人工饲养中国猕猴中SRV、STLV和BV的流行病学调查

非人灵长类动物是十分重要的生物医学资源。由于与人类在生理生化、免疫、遗传等方面近似,猕猴是重要的非人灵长类实验动物之一。然而,猕猴作为自然宿主,易感染D型逆转录病毒(simian type D retrovirus,SRV)和T淋巴细胞白血病病毒(simian T lymphotropic virus,STLV)这两种逆转录病毒,并可能会影响AIDS猕猴动物模型等的研究结果。猴B病毒(ceropithecine herpesvirus1,BV)对猕猴及动物从业人员均有危害。云南省拥有较大规模的中国猕猴繁殖种群。基于以上原因,建立SPF级别的中国猕猴种群十分必要。该文应用PCR技术筛查了人工饲养种群中411只中国猕猴的SRV、STLV和BV感染流行情况。结果表明:SRV、STLV和BV的阳性感染率分别为19.71%(81/411)、13.38%(55/411)和23.11%(95/411)。同时比较分析了不同性别及年龄组中国猕猴的病毒感染情况。该研究将有助于建立SPF级别的中国猕猴繁殖种群。     

Herpes B-virus seroreactivity in a colony of Macaca mulatta: data from the Sabana Seca Field Station, a new specific pathogen-free program

The demand for B-virus-free animals for biomedical research is increasing, while at the same time the availability of such animals is decreasing. The establishment of Specific Pathogen-Free (SPF) breeding macaque colonies is a priority of the National Institutes of Health. Nevertheless, it is well known that seroreactivity to B-virus can be difficult to interpret, particularly as it can vary over time in a single animal. The aim of the present study was to implement a reliable algorithm to examine B-virus reactivity among the rhesus monkey population of the Caribbean Primate Research Center. The sensitivity and specificity of our assay were determined using reports from two different laboratories as references. Whereas 95.4% of animals showed consistent serological status and 4.6% of animals recruited to this SPF program showed serovariability to B-virus over the initial 2 years of examination. Implications for all SPF programs are discussed in this article.     

SNP‐based genetic characterization of the Tulane National Primate Research Center's conventional and specific pathogen‐free rhesus macaque (Macaca mulatta) populations

Background

The rhesus macaque is an important biomedical model organism, and the Tulane National Primate Research Center (TNPRC) has one of the largest rhesus macaque breeding colonies in the United States.

Methods

SNP profiles from 3266 rhesus macaques were used to examine the TNPRC colony genetic composition over time and across conventional or SPF animals of Chinese and Indian ancestry.

Results

Chinese origin animals were the least genetically diverse and the most inbred; however, since their derivation from their conventional forebearers, neither the Chinese nor the Indian SPF animals exhibit any significant loss of genetic diversity or differentiation.

Conclusions

The TNPRC colony managers have successfully minimized loss in genetic variation across generations. Although founder effects and bottlenecks among the Indian animals have been successfully curtailed, the Chinese subpopulation still show some influences from these events.     

Genetic characterization of specific pathogen‐free rhesus macaque (Macaca mulatta) populations at the California National Primate Research Center (CNPRC)

A study based on 14 STRs was conducted to understand intergenerational genetic changes that have occurred within the California National Primate Research Center's (CNPRC) regular specific pathogen‐free (SPF) and super‐SPF captive rhesus macaque populations relative to their conventional founders. Intergenerational genetic drift has caused age cohorts of each study population, especially within the conventional population, to become increasingly differentiated from each other and from their founders. Although there is still only minimal stratification between the conventional population and either of the two SPF populations, separate derivation of the regular and super‐SPF animals from their conventional founders has caused the two SPF populations to remain marginally different from each other. The regular SPF and, especially, the super‐SPF populations have been influenced by the effects of differential ancestry, sampling, and lost rare alleles, causing a substantial degree of genetic divergence between these subpopulations. The country of origin of founders is the principal determinant of the MHC haplotype composition of the SPF stocks at the CNPRC. Selection of SPF colony breeders bearing desired genotypes of Mamu‐A^*01 or ‐B^*01 has not affected the overall genetic heterogeneity of the conventional and the SPF research stocks. Because misclassifying the ancestry of research stocks can undermine experimental outcomes by excluding animals with regional‐specific genotypes or phenotypes of importance, understanding founder/descendent genetic relationships is crucial for investigating candidate genes with distinct geographic origins. Together with demographic management, population genetic assessments of SPF colonies can curtail excessive phenotypic variation among the study stocks and facilitate successful production goals. Am. J. Primatol. 72:587–599, 2010. © 2010 Wiley‐Liss, Inc.     

Evidence for a lentiviral etiology in an epizootic of immune deficiency and lymphoma in stump-tailed macaques (Macaca arctoides).

A retrospective study determined that an epizootic of immune suppression and lymphoma in stump-tailed macaques (Macaca arctoides) that began in 1976 was associated with a horizontally spread lentivirus infection. This conclusion was based on serology, epidemiology, pathology, and virus isolation. The lesions found in the stump-tailed macaques were more compatible with lesions seen in SIV-infected rhesus than those seen in rhesus macaques infected with type D retroviruses. A lentivirus, isolated from a rhesus inoculated with lymph node homogenate from a stump-tailed macaque, was designed SIVstm and was pathogenic for rhesus macaques. The isolate was antigenically related to other SIVs as well as to HIV-1 and HIV-2. Two surviving stump-tailed macaques sent to another colony carried SIVstm latently for at least 7 years and disseminated it throughout that colony.     

A Large‐Scale SNP‐Based Genomic Admixture Analysis of the Captive Rhesus Macaque Colony at the California National Primate Research Center

Some breeding facilities in the United States have crossbred Chinese and Indian rhesus macaque (Macaca mulatta) founders either purposefully or inadvertently. Genetic variation that reflects geographic origins among research subjects has the potential to influence experimental outcomes. The use of animals from different geographic regions, their hybrids, and animals of varying degrees of kinship in an experiment can obscure treatment effects under study because high interanimal genetic variance can increase phenotypic variance among the research subjects. The intent of this study, based on a broad genomic analysis of 2,808 single nucleotide polymorphisms (SNPs), is to ensure that only animals estimated to be of pure Indian or Chinese ancestry, based on both demographic and genetic information, are used as sources of infants for derivation and expansion of the California National Primate Research Center's (CNPRC) super‐Specific Pathogen Free (SSPF) rhesus macaque colony. Studies of short tandem repeats (STRs) in Indian and Chinese rhesus macaques have reported that heterozygosity of STRs is higher in Chinese rhesus macaques than in Indian rhesus macaques. The present study shows that heterozygosity of SNPs is actually higher in Indian than in Chinese rhesus macaques and that the Chinese SSPF rhesus macaque colony is far less differentiated from their founders compared to the Indian‐origin animals. The results also reveal no evidence of recent gene flow from long‐tailed and pig‐tailed macaques into the source populations of the SSPF rhesus macaques. This study indicates that many of the long‐tailed macaques held in the CNPRC are closely related individuals. Most polymorphisms shared among the captive rhesus, long‐tailed, and pig‐tailed macaques likely predate the divergence among these groups. Am. J. Primatol. 74:747‐757, 2012. © 2012 Wiley Periodicals, Inc.     

Frequency of prenatal loss in a macaque breeding colony

An accurate knowledge of the historical incidence of prenatal loss is essential for management of breeding colonies and for performing developmental toxicity studies in nonhuman primates. Data from the California Regional Primate Research Center indoor (timed-mated) and outdoor (random-mated) colonies of rhesus, cynomolgus, and bonnet macaques (Macaca mulatta, M. fascicularis, and M. radiata) were evaluated for a 10 year breeding period from 1984 to 1993. Pregnancy outcome data for the three species of macaques summarized in this report indicate that early pregnancy as well as term are vulnerable periods of gestation in terms of prenatal loss. Prematurity as well as twinning were additionally associated with elevated rates of loss during the prenatal or neonatal period. The incidence of pregnancy failure did not appear to be related to different housing/management conditions (i.e., indoor timed-mated vs. outdoor random-mated), parity, animal handling, shipping, or relocation. Some of the annual fluctuations in abortions could be related to disease outbreaks (e.g., measles, pneumonia) in the colony. These data will be invaluable in planning for research needs which focus on developmental biology and perinatology, and in interpreting the significance of abortions following exposure to experimental agents in small numbers of animals. © 1996 Wiley-Liss, Inc.     

A significant productive in vivo infection of resting cells with simian immunodeficiency virus in a macaque with AIDS

Identifying the cells that can be infected with HIV in vivo and thus potentially persist as latent reservoirs is of high priority. Here, we report the major infected cells in a chronically simian immunodeficiency virus (SIV)‐infected macaque that developed AIDS and encephalitis. A majority of the infected cells were detected as non‐proliferating resting cells. SIV‐infected non‐proliferating resting cells were found to be playing an important role in viral pathogenesis, persistence, or reservoir formation.     

Assessment of genetic management at three specific-pathogen-free rhesus macaque (Macaca mulatta) colonies

Genetic management is required to maintain genetic diversity by minimizing inbreeding and genetic subdivision in colonies of animals bred for biomedical research. Polymorphic short tandem repeat (STR) loci are useful for genetic management because they facilitate parentage assignments, genetic characterization of individuals, and estimates of baseline population genetic parameters. Using highly informative STR loci, we estimated gene diversity and F-statistics to determine the level of genetic heterogeneity and genetic structure of three specific-pathogen-free (SPF) rhesus macaque (Macaca mulatta) colonies. Effective population sizes, variance in male reproductive success, and rate of decrease in genetic variability also were estimated for two of the three colonies. We documented the overall success of genetic management in maintaining genetic diversity in captive colonies. We report that even genetically managed SPF colonies, despite maintaining high and stable levels of gene diversity (over 0.75), are prone to genetic subdivision due to different management strategies, founder effects, genetic isolation, and drift. These processes are accelerated by the high variances in male reproductive success and low adult sex ratios that are typical of captive rhesus macaque breeding groups, both of which reduce the effective population sizes of these groups.     

Chromobacterium violaceum infections in 13 non-human primates

Background Recently, an Indian‐origin macaque was found dead and Chromobacterium violaceum was isolated from the skin wound, and hepatic and pulmonary abscesses. Methods By searching the database, a total of thirteen cases of C. violaceum infection in pigtail macaques (n = 8), rhesus macaques (n = 4), and one baboon were identified from 2001 to 2010 at Tulane National Primate Research Center. Medical records were reviewed for breed, sex, age, clinical findings, treatment, outcome, bacteriology, and gross and histological findings. Results Seven pigtail macaques and one Indian‐origin rhesus macaque died of chromobacterial septicemia. All chromobacterial septicemic pigtail macaques were adult with higher incidence in female. Hepatic abscess and thrombosis were typical findings along with pulmonary abscess and thrombosis, renal venous thromboembolism, and necrosuppurative pleuritis, peritonitis, splenitis, myocarditis, pericarditis, and meningoencephalitis. Skin wound, uterine infection, and oral and respiratory exposure were considered the points of entry for these animals. Conclusions This represents the first report of chromobacteriosis in pigtail, rhesus macaque, and baboon. Our experience suggests that chromobacterial infections may be more common in non‐human primates than previously recognized.     

Factors contributing to spontaneous Enterocytozoon bieneusi infection in simian immunodeficiency virus-infected macaques

Background A cohort of SIV‐infected macaques had been used to investigate the effect of dietary supplement, immune status, SIV/AIDS disease progression and serum micronutrients levels on spontaneous acquisition of Enterocytozoon bieneusi infection in SIV‐infected macaques. Methods Twenty‐four SIV‐infected macaques were randomized into 2 groups. One group received a vitamin/mineral supplementation and a second group received a placebo. Both groups were examined for E. bieneusi infection. Results SIV‐infected macaques were more prone to acquire E. bieneusi with the progression of SIV/AIDS, and the increased shedding of infectious spores was directly associated with decreased CD4 lymphocyte and increased circulating SIV, in both supplemented and unsupplemented groups of animals. Dietary supplementation, body composition factors and serum micronutrients levels however had no association with the acquisition of E. bieneusi infection in these animals. Conclusions Acquisition of E. bieneusi infection is related to SIV disease progression, CD4 counts and viral load but independent of changes in body composition and serum micronutrient levels.     

Detecting signatures of inter‐regional and inter‐specific hybridization among the Chinese rhesus macaque specific pathogen‐free (SPF) population using single nucleotide polymorphic (SNP) markers

Background While rates of gene flow between rhesus and longtail macaque populations near their hybrid zone in Indochina have been quantified elsewhere, this study demonstrates that the inter‐specific introgression is not limited to the Indochinese hybrid zone but is more geographically widespread. Methods Twelve rhesus and longtail macaque populations were analyzed using single nucleotide polymorphic (SNP) loci. Results There is evidence for inter‐specific admixture between Chinese rhesus and mainland longtails, with implications for genetic diversity both in the Chinese super‐SPF population at the California National Primate Research Center and in other primate facilities. Eastern Chinese rhesus appeared more highly derived than western Chinese rhesus, and allele sharing between longtails and Chinese rhesus was not random with regard to geographic distance, but no significant nuclear genetic differences between eastern and western Chinese rhesus were detected among the 245 genic SNPs assayed. Conclusion The implications of this inter‐specific admixture for the use of Chinese rhesus and mainland longtail in biomedical research should be considered.     

Rhesus macaques inoculated with molecularly cloned simian immunodeficiency virus

We have isolated a biologically active molecular clone of simian immunodeficiency virus (SIV), SIVmac 1A11, originally obtained from a rhesus macaque at the New England Regional Primate Research Center. Virus derived from cells transfected with this clone is cytopathic for rhesus peripheral blood mononuclear cells, replicates in cultures of rhesus macrophages, and infects rhesus macaques when inoculated intravenously. Six macaques inoculated with SIVmac 1A11 all became infected and produced antibodies to viral envelope glycoproteins that neutralized virus. Antibodies to viral core proteins were detected in only one animal. No clinical signs of disease were observed throughout 7 months postinoculation.     

太行山猕猴研究进展与展望

猕猴广泛分布于从阿富汗到泰国和中国南部的亚洲地区.分布于中国的猕猴有6个亚种.太行山猕猴为猕猴华北亚种Macaca mulatta tcheliensis,系中国所特有,目前仅分布于河南、山西两省交界的太行山和中条山南端地区,为世界野生猕猴分布最北的种群.自20世纪80年代初期开始,学者们在数量与分布、形态学(包括普通形态学、骨骼系统形态学)、生态学、微观生物学、疾病、实验技术等方面,对太行山猕猴展开了研究.本文对已有的研究成果进行了综述,并提出了今后亟待开展的研究工作,以期能有助于对这一珍稀物种的有效保护.     

Infection with a molecularly cloned SIVsm virus elicits high titer homologous neutralizing antibodies with heterologous neutralizing activity

We have evaluated the homologous and heterologous neutralizing antibody response in a cohort of six Macaca nemestrina infected with the cloned virus SIVsm62d that showed different levels of envelope diversification. Two progressor macaques developed AIDS by 1.5 years post-inoculation and four non-progressors were asymptomatic for 3 years of follow-up. All macaques developed high titers of neutralizing antibodies against homologous SIVsm viruses and intermediate titers against SIVsmB670. Heterologous virus neutralization of SIVmac, SIVmne, and HIV-2 was detected at much lower levels in both progressor macaques; only one of four non-progressors had evidence for broader neutralizing antibody activity. We noted changes in potential N-linked glycosylation (PNG) sites in V1/V2, C2, and V4 that were common to multiple macaques. These results support a model for viral neutralization where heterologous neutralization is, in part, driven by a strong homologous response and may be coupled to changes in PNG sites in envelope.     

Social Behavior of Captive Indochinese and Insular Long-Tailed Macaques (Macaca fascicularis) Following Transfer to a New Facility

There are measurable differences in behavior, physiology, social organization, and geographic distribution within and between various species of macaques. We collected information on the social behavior of captive Macaca fascicularis from Indochina and island populations after they had been transferred to a new environment and new social groups. While some changes in behavior occurred over time, we found no decrease in high levels of agonistic behaviors. We interpret this finding in light of previous research reporting that long-tailed macaques show low levels of habitatuation to novelty and are highly aroused in comparison to other macaque species. We found predictable differences in behavior for males, females, and infants, in which infants played more, females had higher levels of contact proximity to other individuals, and males exhibited more sexual and threat behavior. A comparison of social behavior in long-tailed macaques from different origins indicate that Indochinese macaques are generally less affiliative and Indochinese males are more aggressive than their insular counterparts. Differences among macaque species, and within the fascicularis-group, should be considered in management of captive colonies and when interpreting research data.     

Making the animal model for AIDS research more precise: the impact of major histocompatibility complex (MHC) genes on pathogenesis and disease progression in SIV-infected monkeys

Experimentally infected rhesus monkeys serve as an indispensable animal model to assess the pathogenesis, to validate therapy approaches and to develop vaccination strategies against viral diseases such as AIDS threatening the human population. Upon infection with simian immunodeficiency virus (SIV), a retrovirus closely related to the human immunodeficiency virus (HIV), macaques develop clinical manifestations similar to those of HIV-infected humans. As in humans, the disease course is variable. Polymorphic genes of the major histocompatibility complex (MHC) are required for the initiation and regulation of a specific immune response and represent a major host factor accounting for the differential outcome of infection. During the last few years, our understanding of the structure and function of the rhesus macaque MHC has increased substantially. Functional studies have led to the identification of specific SIV and HIV peptide epitopes presented by rhesus macaque MHC molecules. The subsequent development of MHC class I tetramers has allowed further insight into the cellular immune response following SIV-infection. Detailed studies demonstrated that viral escape mutants are generated during the acute and chronic phase of infection and explain why control of viral replication ultimately fails. Furthermore, particular MHC haplotypes which influence disease progression have been discovered. Thus, MHC-typing can have a prognostic potential. The further elucidation of the rhesus macaque MHC and the search for other relevant genes will remain an important task for future research and will stimulate all immunologically-related investigations in macaques.